Increased colon cancer risk after severe Salmonella infection
Increased colon cancer risk after severe Salmonella infection
Lapo Mughini-Gras 0 1
Michael Schaapveld 1
Jolanda Kramers 0 1
Sofie Mooij 0 1
E. Andra Neefjes-Borst 1
Wilfrid van Pelt 0 1
Jacques Neefjes 1
0 National Institute for Public Health and the Environment (RIVM), Antonie van Leeuwenhoeklaan 9, Bilthoven, the Netherlands, 2 Department of Infectious Diseases and Immunology, Utrecht University , Yalelaan 1, Utrecht , the Netherlands, 3 Division of Epidemiology, the Netherlands Cancer Institute (NKI) , Plesmanlaan 121, Amsterdam , the Netherlands, 4 Department of Pathology, Free University Medical Center (VUmc) , Boelelaan 1117, Amsterdam , the Netherlands, 5 Department of Chemical Immunology, Leiden University Medical Center (LUMC) , Einthovenweg 20, Leiden , the Netherlands
1 Editor: Oliver Schildgen, Kliniken der Stadt KoÈln gGmbH , GERMANY
Background Colon cancer constitutes one of the most frequent malignancies. Previous studies showed that Salmonella manipulates host cell signaling pathways and that Salmonella Typhimurium infection facilitates colon cancer development in genetically predisposed mice. This epidemiological study examined whether severe Salmonella infection, usually acquired from contaminated food, is associated with increased colon cancer risk in humans.
Methods and findings
We performed a nationwide registry-based study to assess colon cancer risk after
diagnosed Salmonella infection. National infectious disease surveillance records (1999±2015)
for Dutch residents aged 20 years when diagnosed with salmonellosis (n = 14,264) were
linked to the Netherlands Cancer Registry. Salmonella-infected patients were
laboratoryconfirmed under medical consultation after 1±2 weeks of illness. These datasets also
contained information on Salmonella serovar and type of infection. Colon cancer risk (overall
and per colon subsite) among patients with a diagnosed Salmonella infection was compared
with expected colon cancer risk in the general population. Data from the nationwide registry
of histo- and cytopathology (PALGA) and Statistics Netherlands (CBS) allowed assessing
potential effects of age, gender, latency, socioeconomic status, genetic predisposition,
inflammatory bowel disease (IBD), and tumor features. We found that compared to the
general population, colon cancer risk was significantly increased (standardized incidence ratio
[SIR] 1.54; 95%CI 1.09±2.10) among patients with Salmonella infection diagnosed <60
years of age. Such increased risk concerned specifically the ascending/transverse colon
(SIR 2.12; 95%CI 1.38±3.09) after S. Enteritidis infection (SIR 2.97; 95%CI 1.73±4.76).
Salmonellosis occurred more frequently among colon cancer patients with pre-infectious IBD, a
known risk factor for colon cancer. Colon tumors of patients with a history of Salmonella
infection were mostly of low grade.
Funding: This work was supported by KWF grant
'Salmonella infections and gallbladder cancer' (NKI
2013-5969); ERC Advanced grant ERCOPE. No
funding bodies had any role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Patients diagnosed with severe salmonellosis have an increased risk of developing cancer
in the ascending/transverse parts of the colon. This risk concerns particularly S. Enteritidis
infection, suggesting a contribution of this major foodborne pathogen to colon cancer
It is estimated that over 20% of the global cancer burden is attributable to infectious agents [
In contrast to virally induced cancers [2±4], bacteria have been largely neglected as factors
contributing to cancer [
], with only a few bacterial infections linked to cancer development to
]. This is best established for Helicobacter pylori in connection with gastric cancer 
and Mucosa-Associated Lymphoid Tissue (MALT) lymphoma [
], and for Salmonella Typhi
and gallbladder carcinoma in chronic typhoid carriers [10±13].
Bacteria may contribute to cancer development through inflammation, induction of DNA
damage by toxins, metabolites, and/or manipulation of host cell signaling pathways during
their infection cycle [
]. For instance, H. pylori has been shown to support gastric cell
transformation by secreting toxin CagA that activates the c-Met receptor and induces signaling
, with chronic inflammation acting as a contributing factor. Bacteria can also alter the cell
biology of the host during the infection cycle, as illustrated by Salmonella species that
manipulate host cell signaling pathways to enforce bacterial uptake, intracellular survival and egress
]. Salmonella secretes effector proteins into host cells that activate the host AKT and ERK
pathways. These pathways are also activated in many cancers, and are essential for
transforming pretransformed cells [
]. Another Salmonella effector AvrA activates host β-catenin
signaling and also promotes colon carcinogenesis in mice [
]. If by virtue of altered host cell
signaling bacteria provide one step towards cancer development , it is conceivable that
bacterial infections would then also increase cancer risk. This would be expected particularly
under conditions of long-lasting infections, where the chance of targeting an already
pre-transformed cell is higher. This has been illustrated for Salmonella Typhimurium infection in
normal and genetically predisposed (APC+/-) mice, with the latter developing colon carcinoma
]. Notwithstanding these findings and the demonstrated cellular localization of AvrA in
inflamed, colorectal tumors and their precursor lesions in both experimental mouse models
and human clinical specimens [
], it remains unclear whether the many Salmonella infections
occurring in the human population constitute a risk factor for colon cancer.
Roughly 93.8 million people are infected with Salmonella species annually [
]. While over
2,500 different serovars of Salmonella enterica subspecies enterica exist, serovars Typhimurium
and Enteritidis are responsible for around 70% of salmonellosis cases in Europe. As for many
enteric infections, most salmonellosis cases are not reported, as they generally present with
mild and self-limiting symptoms requiring no medical attention. Consequently, it is estimated
that for every salmonellosis case reported to the national infectious disease surveillance
systems in Europe, approximately 57 cases go unreported [
With 694,000 deaths in 2012, colon cancer is a major cause of cancer morbidity and
mortality worldwide [
], especially for older patients, as colon cancer incidence increases markedly
after the age of 60 years [
]. In 2014, 10,319 residents in the Netherlands were diagnosed
with colon cancer and 3,682 residents died due to colon cancer . Factors predisposing for
colon cancer include Inflammatory Bowel Disease (IBD) [
] and genetic mutations [28,
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29]. Colon cancer incidence increases over the years as a function of largely unknown risk
]. To address whether Salmonella infections constitute yet another risk factor for
colon cancer, we compared the incidence of colon cancer among Dutch residents with a
reported history of Salmonella infection to that in the general Dutch population. Moreover, we
examined potential effects of gender, age, latency, socioeconomic status (SES), type of
infection, IBD, genetic predisposition and tumor pathological features on the association between
Salmonella infection and colon cancer.
The researchers obtained written permission to use and link the different data sets after
anonymization. The contract numbers for the different data sets are: NCR (K16.147 and K15.257)
and PALGA (lzv2016-72).
We performed a retrospective cohort study based on three linked registries described in detail
elsewhere [32±34]. The first registry is maintained by the Dutch National Institute for Public
Health and the Environment (RIVM) and contains national surveillance data on reported
human salmonellosis cases from the Netherlands' laboratory surveillance network, which
includes 16 regional public health laboratories covering 64% of the general Dutch population
]. At the time of analysis, this database included a total of 28,117 unique records of
patients with a laboratory-confirmed Salmonella spp. infection in the Netherlands diagnosed
between January 1999 and December 2015 with associated metadata, i.e. birth date, gender,
date of diagnosis, residence location, isolated Salmonella serovar, and type of infection (enteric,
septicemic, other) (S1 Table). The second database, maintained by the Dutch Association of
Comprehensive Cancer Centers (IKNL), derives from the Netherlands Cancer Registry
(NCR). This registry covers all Dutch residents, the data are more than 95% complete, and
includes 140,685 patients diagnosed with colon cancer (ICD-O-3 codes: C180-C189) in 1999±
2015. These data also include the colon subsite (ascending, transverse, and
descending/sigmoid) in which the tumor has been diagnosed. The third database (Dutch Nationwide
Network and Registry of Histo- and Cytopathology, PALGA) contains the pathology records of all
patients in the Netherlands and is a countrywide database since 1991 .
Data anonymization and linkage
Statistics Netherlands (CBS) acted as a trusted third party for data anonymization and linkage
by adding a Record Identification Number (RIN) as unique identifier for each individual in all
databases. Birth date, gender, residence location, and date of registration formed the basis for
the derivation of the RIN numbers. To this end, CBS used a reference database containing all
mutations due to death or relocation in the Dutch population since 1995, including a complete
housing history of all Dutch residents. After the RIN numbers were added, all personal
identifiers were removed. Based on RIN numbers, patients with a reported Salmonella infection in
the RIVM database were linked to the NCR data on patients with diagnosed colon cancer. The
RIN numbers also allowed coupling to other CBS information, such as the date of death and
the standardized household income (or ªspendable incomeº, adjusted for the size and
composition of the household) used as a proxy for SES. The linkage with the PALGA data first
required de-anonymization by CBS and then transfer of the data to IKNL before transfer to
PALGA for record classification. To this end, the colon cancer patient group with a reported
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Salmonella infection (n = 65) was mixed with a three-fold larger randomly selected age- and
gender-matched group of colon cancer patients without such infection history (n = 194). Their
pathology records were then provided to the pathologist for further classification, without
information on infection. The pathologist then reported the classifications to CBS for
anonymization and addition of RIN numbers before releasing the data for analysis.
Participants and classification
All data sets were cleared from duplicates. Patients with the reporting date of Salmonella
infection falling before the start (January 1st, 1999) or after the end (December 31st, 2015) of the
study period were censored. After linking the records of the 28,117 salmonellosis cases in the
RIVM database to those of the 140,685 colon cancer patients in the NCR database, 227
matches (i.e. salmonellosis cases with a diagnosed colon cancer) were found, whereas 27,890
salmonellosis cases did not have a diagnosed colon cancer. In the main analysis, the follow-up
period expressed in years at risk started one year after the date of Salmonella infection and
ended at the date of colon cancer diagnosis, date of death, or end of the study period,
whichever came first. We excluded 103 salmonellosis cases diagnosed with colon cancer before
Salmonella infection and 28 salmonellosis cases having colon cancer diagnosed <1 year from
Salmonella infection. Patients younger than 20 years at Salmonella infection (n = 12,008)
were also excluded as their risk of colon cancer is virtually zero (no colon cancer cases were
observed in this age group after matching with Salmonella data). Finally, all 1,714 salmonellosis
cases without a diagnosed colon cancer that could be followed for less than one year after
infection were removed. For patients with multiple Salmonella infections reported over time,
only the first one was considered. Our final cohort therefore comprised 14,264 salmonellosis
cases aged 20 years when diagnosed with Salmonella infection between 1999 and 2015, and
with at least one year follow-up post-infection. Of these, 96 were subsequently diagnosed with
colon cancer 1 year after the reported Salmonella infection (Fig 1).
As colon cancer incidence increases steeply after 60 years of age due to several factors [
] (S1 Fig) that may in principle dilute the hypothesized effect of Salmonella infection, we
also performed additional analyses considering only those patients younger than 60 years at
infection. For sensitivity analysis of the follow-up period, additional analyses were performed
with the time at risk starting at 4, 7 or 10 years after infection. As regard to the final PALGA
data set for analysis, this comprised 65 colon cancer patients with a reported Salmonella
infection and 194 age- and gender-matched colon cancer patients without a reported history of
Salmonella infection to be used as control group.
In the main analysis, time at risk started one year after diagnosis of salmonellosis and ended at
the date of colon cancer diagnosis, date of death or January 1st, 2016, whichever occurred first.
Colon cancer risk among salmonellosis cases was first estimated as compared with colon
cancer risk in the Dutch population (i.e. the baseline reference risk) by calculating standardized
incidence ratios (SIRs) of colon cancer (overall and per colon subsite) by dividing the observed
number of colon cancer patients among those with a reported Salmonella infection by the
expected number of colon cancers based on subsite-, age-, gender- and calendar year-matched
colon cancer incidence rates in the general Dutch population, as derived from NCR figures.
SIRs for colon cancer overall and per colon subsite (ascending, transverse, or
descending/sigmoid) were then stratified by gender, age at infection (20±39, 40±49, 50±59, 60±69, 70
years), follow-up time in years at risk after infection (1±7 and >7 years, according to median
follow-up), diagnosed Salmonella serovar (Typhimurium and its monophasic variant with
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Fig 1. Schematic representation of the data management process. Number of patients in the two linked databases, as well as the number of patients excluded
from further analyses according to each inclusion criterion.
antigenic formula 1,4,[
],12:i:-, Enteritidis, or others), and type of infection (enteric,
septicemic, or others like urinary tract or wound infections). To increase statistical power and
because the risk of cancer in the ascending and transverse colon was comparable, these two
adjacent colon subsites were combined. 95% confidence intervals (95%CIs) for SIRs were
calculated assuming a Poisson distribution. Tests for heterogeneity and trends in SIRs were
performed using Poisson regression analysis of collapsed person-time data.
Joint Cox proportional hazards regression analysis with attained age as time-scale,
accounting for death as competing risk, with entry into the at-risk period one year after the age at
Salmonella infection, was then used to assess associations of gender, age group at infection,
infecting Salmonella serovar, type of infection and SES (low vs. high, according to median
standardized household income) with cancer risk in the ascending/transverse colon and
descending/sigmoid colon simultaneously within the cohort. In this analysis, only the patients with
reported salmonellosis were included, meaning that no increased or decreased risk for colon
cancer after Salmonella infection can be shown by the within-cohort analysis, as there is no
baseline reference risk (i.e. colon cancer incidence in the general population) to be used for
comparison. The purpose of within-cohort comparisons was therefore to assess whether there
were also differences in cancer incidence in the different portions of the colon altogether as a
function of age, gender, serovar, infection type and SES in the salmonellosis patients alone.
Proportional hazard assumptions were verified using graphical and residual-based methods.
Associations were expressed as adjusted hazard ratios (HRs) and corresponding 95%CIs.
An additional analysis assessed potential effects of IBD, genetic predisposition (mutations
in the Ras/Raf/Mapk pathway), microsatellite instability (MSI), tumor stage (0-I, II, III, IV,
based on TNM classification) and differentiation in colon cancer patients who experienced
salmonellosis vs. those who did not. This analysis was performed using multivariable binomial
regression models, which in the group of colon cancer patients for which the PALGA records
were assessed (n = 259) estimated the risk of having had salmonellosis (binary response) as a
function of IBD, genetic predisposition, MSI, tumor stage and differentiation, with correction
for the matching variables (age and gender). Associations were expressed as adjusted risk
ratios (RRs) and corresponding 95%CIs. Retained in the models were only those factors that
were significantly associated with the outcome (i.e. having experienced reported Salmonella
infection) or that changed the RRs of the other covariates >10% when removed from the
model. In all analyses, p-values <0.05 were considered statistically significant. Statistical
analysis was performed using STATA 14 (StataCorp LP, College Station, USA).
Description of the cohort
Our cohort comprised 14,264 patients (53.6% women and 46.4% men) aged 20 years when
diagnosed with Salmonella infection between 1999 and 2015 and with at least 1 year follow-up
post-infection (Fig 1). The median age at Salmonella infection was 46 years (interquartile
range [IQR], 29±63), with 70.5% being <60 years at infection. Median follow-up was 7 years
(IQR, 3±12). In total, 96 patients were diagnosed with colon cancer 1 year after the reported
Salmonella infection (Fig 1), with a median time between salmonellosis and colon cancer
diagnoses of 5 years (IQR, 3±9). At 61 and 91 years of age, cumulative incidence of colon cancer
among salmonellosis cases was 1.31% (95%CI, 0.784±1.84%) and 4.83% (95%CI, 3.89±5.79%),
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respectively (S2 Fig). Of the 14,264 Salmonella infections included in the cohort, 89% were
enteric infections, 5% were septicemic infections, and 6% were of other types.
Colon cancer risk after Salmonella infection as compared to the population
Colon cancer risk among salmonellosis patients was estimated as compared with the general
Dutch population (baseline) by calculating SIRs and corresponding 95%CIs based on colon
subsite-, age-, gender- and calendar year-matched colon cancer incidence rates. Compared to
the population, cumulative incidence of colon cancer among salmonellosis patients was
slightly higher until 70 years of age (Fig 2, inset), but overall colon cancer incidence among
salmonellosis patients was not significantly increased (SIR 1.17; 95%CI 0.95±1.43) (Table 1).
When the different Salmonella serovars were considered, cumulative incidence of colon
cancer for S. Enteritidis-infected patients was higher than in the population (Fig 2). When
considering only patients under 60 years of age at the time of Salmonella infection, colon cancer
risk was significantly increased (SIR 1.54; 95%CI 1.09±2.10) (Table 1), and such risk further
increased when considering patients infected with S. Enteritidis (SIR 1.86; 1.17±2.82)
(Table 2). SIRs did not differ over gender (Table 1).
Fig 2. Cumulative incidence of colon cancer in patients with Salmonella infection and in the general population. Cumulative incidence of colon cancer over attained
age in patients with a reported history of Salmonella infection and in the general population. Inset: cumulative incidence of colon cancer in patients with any Salmonella
serovar infection and in the general population. Main graph: cumulative incidence of colon cancer in patients infected with the two major Salmonella serovars (Enteritidis
and Typhimurium), the other less often diagnosed Salmonella serovars combined, and in the general population.
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§3 colon cancer cases were excluded from the colon subsite-specific analysis as they had cancer involving both the ascending/transverse and descending/sigmoid regions
of the colon.
Risk of colon cancer as a whole and per subsite by gender and age at Salmonella infection for patients of all ages ( 20 years) and for those <60 years at infection.
Observed (Obs) and expected (Exp) numbers of cancers, standardized incidence ratio (SIR) with 95% confidence interval (CI), test of SIR for heterogeneity and trend.
The colon is morphologically different at subsites, with the ascending and transverse parts
being most exposed to Salmonella infection, as they are closest to the terminal ileum where
such infections mainly reside. For this reason, the analysis was stratified by colon subsite
(ascending/transverse or descending/sigmoid colon) showing that salmonellosis patients had
significantly increased SIRs of cancer in the ascending/transverse colon, but not in the
descending/sigmoid colon, as compared to the general population; this was consistent over
gender, age, and follow-up periods (Tables 1 and 2). SIR of cancer in the ascending/transverse
colon was 1.48-fold (95%CI 1.14±1.88) increased, and over 2-fold increased (SIR 2.12; 95%CI
1.38±3.09) for patients infected before 60 years of age. Age stratification showed that SIRs of
cancer in the ascending/transverse colon among salmonellosis cases decreased to population
levels at 70 years of age when infected (Table 1). There was no effect of follow-up duration:
the SIRs of cancer in the ascending/transverse colon among patients infected before 60 years
were 2.22 (95%CI 1.11±3.96) and 2.05 (95%CI 1.15±3.38) at 1±7 and >7 years post-infection,
respectively (Table 2).
The two most common serovars, S. Typhimurium and S. Enteritidis, differ in
epidemiological and biological aspects, which could lead to different risks for colon cancer. The other
Salmonella serovars were collectively analyzed. Cumulative incidence of colon cancer was highest
for patients infected with serovar Enteritidis (Fig 2, main graph), especially when considering
the ascending and transverse parts of the colon (Fig 3, main graph) unlike the descending and
sigmoid parts of the colon (S3 Fig).
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§3 colon cancer cases were excluded from the colon subsite-specific analysis as they had cancer involving both the ascending/transverse and descending/sigmoid regions
of the colon.
²Salmonella isolated from urinary tract or wound infections.
Risk of colon cancer as a whole and per subsite by follow-up time, infecting Salmonella serovar and type of infection for patients of all ages ( 20 years) and for those
<60 years at infection. Observed (Obs) and expected (Exp) numbers of cancers, standardized incidence ratio (SIR) with 95% confidence interval (CI), test of SIR for
The increased SIR of cancer in the ascending/transverse colon after S. Enteritidis infection
was as high as 2.97 (95%CI 1.73±4.76) when infection was diagnosed before 60 years of age
(Table 2), and ever higher (3.22; 95%CI 1.84±5.23) for patients aged 40±59 years at infection
(Table 3). Regarding the type of Salmonella infection, significantly increased SIRs were only
observed among enteric infections (Tables 2 and 3).
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Fig 3. Cumulative incidence of cancer in the ascending and transverse parts of the colon. Cumulative incidence of cancer in the ascending/transverse colon over
attained age in patients with a reported history of Salmonella infection and in the general population. Inset: cumulative incidence of cancer in the ascending/transverse
colon in patients with any Salmonella serovar infection and in the general population. Main graph: cumulative incidence of cancer in the ascending/transverse colon in
patients infected with the two major Salmonella serovars (Enteritidis and Typhimurium), the other less often diagnosed Salmonella serovars combined, and in the general
The time to onset of colon cancer after Salmonella infection is unknown and we therefore
repeated the analyses considering the time at risk to start at 4, 7 or 10 years post-infection. The
increased SIRs of cancer in the ascending/transverse colon after Salmonella (Enteritidis)
infection occurring between 20 and 60 years of age remained significant over the analyses (S2±S7
Within-cohort comparisons and analysis of pathology records
Joint Cox analysis showed that, within the cohort (i.e. among salmonellosis cases only), the
risk of colon cancer did not differ significantly by sex, serovar, age at infection, type of
infection or SES, and that the HRs of cancer in the ascending/transverse colon vs.
descending/sigmoid colon within the cohort were not significantly different, possibly because of low
statistical power (S8 Table).
From the National Pathology Records data base PALGA, information was obtained on
IBD, mutations in the Ras/Raf/Mapk pathway, MSI, tumor stage and differentiation for 65
colon cancer patients with prior salmonellosis and this was compared with 194 age- and
gender-matched colon cancer patients without a reported history of Salmonella infection. Colon
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§3 colon cancer cases were excluded from the colon subsite-specific analysis as they had cancer involving both the ascending/transverse and descending/sigmoid regions
of the colon.
²Salmonella isolated from urinary tract or wound infections.
Risk of colon cancer as a whole and per subsite following an infection with Salmonella Enteritidis stratified by age at infection, type of infection, and by follow-up time
for patients of all ages ( 20 years) and for those <60 years at infection. Observed (Obs) and expected (Exp) numbers of cancers, standardized incidence ratio (SIR) with
95% confidence interval (CI), test of SIR for heterogeneity.
cancer patients with reported salmonellosis more frequently had IBD (7.7%) than colon cancer
controls (2.1%, RR 3.1; 95%CI 1.2±8.0) (Table 4 and S9 Table).
IBD patients represented a small group (n = 5) of all patients with a salmonellosis history
and these were diagnosed with IBD before salmonellosis (Table 5). Colon cancer patients with
a history of Salmonella infection were also significantly more likely to present with lower stage
tumors (Table 4 and S9 Table). Occurrence of genetic predisposition, MSI and
undifferentiated cancer phenotypes did not differ between colon cancer patients with and without
reported history of salmonellosis (S9 Table).
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§ All estimates are corrected for the matching variables gender and age at cancer diagnosis. Retained in the models are only those factors that were significantly
associated with the outcome (i.e. having experienced reported Salmonella infection) or that changed the RRs of the other covariates >10% when removed from the
model. Besides inflammatory bowel disease (IBD) and tumor stage (based on TNM classification), variables tested for association in the analysis were presence/absence
of microsatellite instability (MSI), genetic predisposition (mutations in the Ras/Raf/Mapk pathway) and tumor differentiation (S9 Table).
Output of the multivariable binomial regression analysis predicting reported Salmonella infection among patients with colon cancer as a function of genetic and tumor
pathological factors of the colon cancer patients using a three times larger gender- and age-matched colon cancer control group without reported Salmonella infection.
Colon cancer cases per colon subsite affected with (Salm+) and without (Salm-) a reported Salmonella infection, risk ratio (RR) and 95% confidence interval (CI).
To assess whether Salmonella infection constitutes a risk factor for colon cancer, we compared
the incidence of colon cancer among Dutch residents with a history of (severe) Salmonella
infection to that in the general Dutch population (i.e. the baseline reference incidence).
Moreover, we examined potential effects of gender, age, latency, serovar, type of infection, SES, IBD,
genetic predisposition and tumor pathological features on the association between Salmonella
infection and colon cancer.
We observed an increased risk of cancer in the ascending and transverse parts of the colon
in patients with a reported history of Salmonella infection, with the highest risk for those
patients aged <60 years at the time of infection, as compared to the general population. This
increased risk was most strongly related to infection with S. Enteritidis. While the terminal
ileum is where Salmonella mainly resides [
], the ascending and transverse portions of the
Year of diagnosis for inflammatory bowel disease (IBD), Salmonella infection, and colon cancer in patients with a history of Salmonella infection. The data are extracted
from the pathology records (via PALGA) of the selected patient group.
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colon are colon subsites most exposed to Salmonella bacteria leaving the ileum. Various factors
may then contribute to carcinogenesis in the colon, including inflammation, dysbiosis and
continuous growth of epithelial cells with a risk of acquiring pretransforming mutations.
Consequently, pre-malignant forms (i.e. polyps) of colon cancer are often observed, especially at
higher age. If Salmonella infection provides one step in the multistep process resulting in
], infection of premalignant colon polyps could induce full transformation.
We also assessed several potential confounders, such as SES, IBD, genetic predisposition
and tumor pathological features. SES is a proxy for many factors, including diet, general health
status, smoking behavior, physical activity and obesity [
], which did not contribute
significantly to salmonellosis-associated colon cancer risk. Only IBD occurrence differed
significantly between colon cancer patients with and without reported Salmonella infection, but the
small number of IBD patients (n = 5) relative to the total number of colon cancer patients with
a history of salmonellosis for which IBD status was known (n = 65), suggests that IBD
contribution is minimal. Yet, IBD may predispose to a more intense or longer periods of Salmonella
infection, thereby further increasing the risk of developing colon cancer.
To date, several risk factors for colon cancer have been identified. These include IBD [
] and genetic mutations [
]. Yet colon cancer incidence increases over the years as a
function of largely unknown risk factors [
]. Whether colon cancer is pure `bad genetic
luck' or whether external factors like a protein- and fat-rich diet and sedentary lifestyle are
essential contributors, is still unclear . Fruit, for example, contains anti-oxidants that may
reduce radical formation and DNA mutations leading to colon cancer [
]. Dietary calcium,
vitamin D and folate can also modulate colon carcinogenesis [
]. Moreover, both chronic
inflammation (e.g. IBD) and diet may affect the gut microbiome, and some microorganisms
may secrete mutating compounds directly [
] or indirectly by modulating the immune
response in the colon [
]. While genetic predisposition (SIR 2.2±3.9) and IBD (SIR 2.6±2.8)
constitute the major reported factors, alcohol abuse, obesity, and consumption of red and
processed meat (SIR 1.2) contribute more marginally. Obviously, consumption of (undercooked)
meat is also a risk factor for acquiring Salmonella infection [
], which may also contribute to
this association with colon cancer. Moreover, cancer is fueled by deregulation of signaling
pathways in control of cellular growth and proliferation, and these pathways are sometimes
targeted by bacteria to establish infection [
]. Trivializing a major foodborne pathogen like
Salmonella as a mere causative agent of gastroenteritis would therefore ignore its known
potential to manipulate host cell signaling pathways in their infectious cycle [
] in a way
that would also contribute a step in the cancer formation cascade. The role of bacterial
effectors in host cell cell biology is complex. For example, Salmonella protein AvrA is injected in
host cells by the Type Three Secretion System (TTSS) to suppress apoptosis particularly in the
context of enteropathogenic salmonellosis to prolong intracellular bacterial survival [
This would explain why infection invasiveness does not associate to transformation, as
significantly increased SIRs for colon cancer were only observed for enteric, and not for septicemic,
infections. AvrA-expressing Salmonella bacteria also increase the number of stem cells and
proliferative cells in infected intestinal mucosa by activating the Wnt/β-catenin pathway [
with Salmonella being confirmed to persist in the colon for up to 45 weeks, not only in
epithelial cells on the colonic luminal surface and base of the crypts, but also in invading colorectal
tumors whose incidence has been reported to be significantly increased in AvrA+ vs. AvrA−
Salmonella infected mice [
]. This is not only the case in mice and AvrA is also detected in
inflamed colorectal tumors and its precursor lesions in human clinical specimens [
providing at least one plausible mechanistic explanation as to how Salmonella infections could
contribute to colon cancer development.
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We observed that the tumors of patients with a history of Salmonella were mainly of low
grade. It is unclear why, but one option is that, by virtue of their history, these tumors are
different from those developing without a contribution from Salmonella infection, as observed
for the gallbladder carcinomas with a history of S. Typhi infection [
]. The tumors associated
with salmonellosis may have started from already pre-transformed cells, and while
transformation from a pre-malignant to a malignant (full and advanced carcinoma) state would usually
take around 4 years, cell, organoid and mouse experiments suggest that Salmonella infection
can accelerate this transformation considerably [
]. Yet, it is unknown how long this
transformation would take in humans. For this reason, we repeated the analyses setting the start of the
follow-up period at 1, 4, 7 and 10 years post-infection, which all yielded similar increased risks
of colon cancer after S.Enteritidis infection. It is possible that the patients diagnosed with
colon cancer within 4 years from Salmonella infection had already premalignant colon polyps
or adenomas whose transformation to cancer was accelerated by the Salmonella infection itself,
thereby mimicking the situation observed under laboratory conditions [
], whereas patients
diagnosed with colon cancer afterwards have had a different contribution from Salmonella
infection, possibly including induction of the pre-transformed state itself.
S. Enteritidis infection showed a 3-fold increased risk of cancer in the ascending/transverse
colon as compared to the general population, although it was not significantly different from
the effects of the other Salmonella serovars when considering the salmonellosis cases only (i.e.
within the cohort). There are several major differences in the epidemiology and biology of
Salmonella serovars that could potentially explain any serovar effect on colon cancer risk. For
instance, S. Typhimurium, unlike S. Enteritidis, contaminates virtually all foods of animal
origin (mainly meat products), vegetables and even the environment. People are thus repeatedly
exposed to S. Typhimurium from a young age [
] and may acquire some immunity to
(severe) infection with this serovar. By contrast, S. Enteritidis is a highly poultry-adapted
serovar whose transmission in industrialized countries like the Netherlands is almost exclusively
related to consumption of raw eggs and other poultry products [
], affecting mainly
adults. Consequently, acquired immunity against S. Enteritidis is less likely to occur and
infection may therefore be more severe or persistent. It is also possible that one or more of the 29
effectors specific for serovar S. Enteritidis would support transformation more incisively .
The molecular mechanisms of the different Salmonella serovar effects on colon tumor
formation are as yet unclear.
Laboratory experiments indicate that Salmonella (Typhi and Typhimurium) can contribute
to some steps in the multistep process to oncogenic transformation [
]. A coincidental
infection of a pre-transformed cell may therefore suffice in driving cancer development. Persistent
or severe Salmonella infections would then increase the risk of developing cancer, as the
chances of infecting a pre-transformed cell are higher under these circumstances. Patients
diagnosed with Salmonella infections and reported to public health authorities in the
Netherlands are typically (severely) ill for at least 1±2 weeks. This represents only a small part of all
Salmonella infected persons. The number of symptomatic Salmonella infections in the Dutch
population during 1999±2015 is estimated at around 994,200 [
], approximately 35 times
higher than the diagnosed cases included in this study. Most infections have a mild clinical
presentation, with symptoms lasting only a few days, thus prompting no medical attention,
laboratory testing and reporting. Consequently, the group of colon cancer patients without a
reported Salmonella infection may have included patients with undiagnosed, milder
Salmonella infections, with unknown contribution to colon cancer. Of the circa 93.8 million cases of
salmonellosis estimated to occur annually worldwide, a significant number is attributable to S.
], which may thus in principle contribute to colon cancer formation in a
significant number of persons. The Salmonella infections included in this study represent severely
14 / 19
infected patients diagnosed after long-lasting illness, including severe symptoms like bloody
diarrhea, dehydration, etc. requiring medical attention and even hospitalization. The potential
contribution to colon cancer of the larger number of unreported (mild) Salmonella infections
occurring in the population is implicitly included in the baseline colon cancer incidence used
for comparison, so the increased risks identified here can be considered a conservative
estimate. This is further suggested by serology studies suggesting that subjects with increased
Salmonella FliC antibody titers may also have an increased risk of colorectal cancer [
This study was inspired by an experiment showing that APC+/- mice infected with S.
Typhimurium developed colon cancer following infection [
]. This does not mean that the
epidemiological results reported here can be explained by these mouse experiments. Indeed,
the pathogenesis of salmonellosis in mice differs from humans. Yet, by virtue of the ability of
Salmonella bacteria to manipulate host cell signaling pathways in a way that would promote
their transformation, one of the possible outcomes of Salmonella infections could in fact be
similar regardless of the host in question. Human are not germ-line APC heterozygous, which
would decrease risk of transformation by Salmonella. Yet, humans have a considerably larger
colon than mice, and more cells±including pretransformed cells- can be infected. It was
unclear how these factors weight in the ultimate outcome of infection and transformation, but
an increased risk of colon cancer is observed at least following S.Enteritidis infection. While
the results of this epidemiological study suggest that Salmonella infection increases colon
cancer risk and mechanistic biological explanations are available [
], the exact cascade of
events leading to such increased risk needs to be further disentangled.
In conclusion, to a brief list of bacterial pathogens contributing to cancer, including so far
S. Typhi infection in association with gallbladder cancer (SIR 7±11) [
10, 11, 13
] and H. pylori
infection with gastric cancer (SIR 5.8) [
], our data suggest that the major foodborne
pathogen Salmonella Enteritidis in association with colon cancer may be added. While we have
described a molecular mechanism for the role of Salmonella in gallbladder carcinoma
formation in man and colon cancer formation in mice [
], these data have to be confirmed in the
human colon cancer samples in patients with a history of severe Salmonella infection.
Independent confirmation of the relationship between S.Enteritidis and colon cancer in independent
data sets will further strengthen these points, as required to convince health authorities in
allowing early participation of patients diagnosed for this pathogen into National Colon
Cancer Screening Programs.
S1 Table. Description of all patients with a reported Salmonella infection in the
Netherlands during 1999±2015 (n = 28,117).
S2 Table. Colon cancer risk by gender and age at Salmonella infection, with time at risk
starting 4 years after infection.
S3 Table. Colon cancer risk by follow-up, Salmonella serovar and type of infection, with
time at risk starting 4 years after infection.
S4 Table. Colon cancer risk by gender and age at Salmonella infection with time at risk
starting 7 years after infection.
15 / 19
S5 Table. Colon cancer risk by follow-up, Salmonella serovar and type of infection, with
time at risk starting 7 years after infection.
S6 Table. Colon cancer risk by gender and age at Salmonella infection with time at risk
starting 10 years after infection.
S7 Table. Colon cancer risk by follow-up, Salmonella serovar and type of infection, with
time at risk starting 10 years after infection.
S8 Table. Joint Cox proportional hazards analysis of colon cancer within the cohort.
S9 Table. Outputs from the univariate binomial regression analysis of pathology records.
S1 Fig. Incidence of colon cancer according to age in the Dutch population between 1999
and 2016 (in bars) and projected standardized incidence ratio (SIR) and 95% confidence
interval (CI) for colon cancer among those infected in the same decade of life with
S2 Fig. Cumulative incidence of colon cancer by attained age in patients with reported
S3 Fig. Cumulative incidence of cancer in the descending and sigmoid parts of the colon.
We thank Ivo Gorissen from Netherlands Statistics (CBS), Vincent Ho from the Dutch
Association of Comprehensive Cancer Centers (IKNL), and Valery Lemmens from PALGA, the
nationwide network and registry of histo- and cyto-pathology in the Netherlands, for support
with database linking and patient anonymization. We further thank Ilana Berlin, Frits
Rosendaal and Wim Fibbe for a critical reading of the manuscript. We also thank the participating
Regional Public Health Laboratories (RPHL) for their contribution to the data collection.
Requests to enter datasets can be addressed to Dr Lapo Mughini-Gras, National Institute
for Public Health and the Environment (RIVM), Antonie van Leeuwenhoeklaan 9, 3721 MA
Bilthoven, the Netherlands. Email: .
Conceptualization: Lapo Mughini-Gras, Michael Schaapveld, Wilfrid van Pelt, Jacques
Data curation: Lapo Mughini-Gras, Michael Schaapveld, Jolanda Kramers, Sofie Mooij.
Formal analysis: Lapo Mughini-Gras, Michael Schaapveld, Jolanda Kramers, Sofie Mooij, E.
Andra Neefjes-Borst, Wilfrid van Pelt.
Funding acquisition: Jacques Neefjes.
Investigation: Jolanda Kramers, E. Andra Neefjes-Borst.
16 / 19
Methodology: Lapo Mughini-Gras, Sofie Mooij, Wilfrid van Pelt.
Supervision: Wilfrid van Pelt, Jacques Neefjes.
Writing ± original draft: Jacques Neefjes.
Writing ± review & editing: Lapo Mughini-Gras, Michael Schaapveld, Jolanda Kramers, Sofie
Mooij, E. Andra Neefjes-Borst, Wilfrid van Pelt, Jacques Neefjes.
17 / 19
18 / 19
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